Autoreactive T cells that are capable of inducing disease exist in normal adult animals, but are maintained in a dormant or inactive state due to the suppressive functions of regulatory T cells (Treg). We have demonstrated that regulatory T cells can be easily identified in normal lymphoid tissues by expression of CD4, the interleukin-2 receptor alpha chain (CD25), and the transcription factor, FoxP3. Transfer of CD4+CD25-Foxp3- T cells to immunoincompetent mice results in the development of autoimmune disease that can be prevented by co-transfer of CD4+CD25+Foxp3+ T cells. The major goals of this project are to define the function and mechanism of action of Treg cells in vivo. We have used both polyclonal Treg and Treg that have been induced in vitro by stimulation of naive T cells in the presence of TGF-beta. TGF-beta induced Tregs (iTregs) have many of the phenotypic features of thymic-derived Tregs (nTregs), as they are anergic, suppressive, and can prevent the development of autoimmune disease. Furthermore, they can be generated from any naive antigen specific CD4+Foxp3- cell in vitro in unlimited numbers. We have analyzed the in vivo dynamics of the interaction between polyclonal Foxp3+ Treg, effector T cells (Teff), and DC in order to further our understanding of the mechanisms of Treg-mediated suppression. Co-transfer of polyclonal activated Treg into normal mice attenuated the induction of experimental autoimmune encephalomyelitis. Suppression of disease strongly correlated with a reduced number of Teff cells in the spinal cord, but not with Treg-mediated inhibition of Th1/Th17 differentiation. Co-transfer of Treg with TCR transgenic Teff cells followed by immunization by multiple routes resulted in an enhanced number of Teff cells in the lymph nodes draining the site of immunization without an inhibition of Teff differentiation. Fewer Teff cells could be detected in the blood in the presence of Tregs and fewer T cells could access a site of antigen exposure in a modified delayed type hypersensitivity assay. Teff cells recovered from LN in the presence of Tregs expressed decreased levels of CCR4, syndecan, and the sphingosine phosphate receptor, S1P1. Thus, polyclonal Tregs influence the Teff cell responses by targeting trafficking pathways, thus allowing immunity to develop in lymphoid organs, but limiting the number of potentially auto-aggressive cells that are allowed to enter tissues. The biologic effects of antigen-specific Tregs in vivo were dramatically different frm the nTregs. To analyze the function of antigen-specific iTregs in vivo, we used a cell transfer system very similar to the one developed for the analysis of polyclonal Treg function in vivo. We generated iTregs from CD4+Foxp3- T cells derived from TCR transgenic (Tg) mice on a RAG deficient background by stimulation with plate-bound anti-CD3 and anti-CD28 in the presence of TGF&#8722;beta and IL-2. We first co-transferred the marked iTreg together with congenically marked, CFSE-labeled nave T cells from the same TCR Tg donor to a normal recipient and immunized the recipient with the target peptide in CFA or IFA. 4-5 day later, we determined the state of activation, differentiation, and expansion of the T effector (Teff) in the draining LN. In marked contrast to the results obtained with polyclonal nTreg, the expansion of the nave Teff was inhibited by >90% either when measured by the total percentage of donor Teff in the draining LN, by CFSE dilution or when the absolute number of Teff cells was counted. The small number of Teff cell recovered from the draining node failed to upregulate CD44 expression indicating that their activation was also markedly impaired. Furthermore, when the recovered Teff cells were re-stimulated with PMA/ionomycin to measure cytokine production, expanded Treg from mice that had not received iTregs produced both IFN-gamma and IL-17, while the few recovered T cells from mice iTreg treated mice failed to produce either of these cytokines. Thus, iTregs profoundly inhibited all aspects of the T cell activation cascade in vivo. We could also perform these studies using congenically marked iTreg. In contrast, to the complete lack of cell expansion of the Teff in iTreg-treated animals, iTregs from these mice readily proliferated as measured by CFSE dilution. The profound inhibition of Teff activation suggested that the iTreg were inhibiting antigen presentation by acting on DC.. One possibility is that the iTreg killed DC by utilizing the perforin-Granzyme B pathway as nTregs have been shown to inhibit antigen presentation by activated B cells by this pathway in vitro. However, iTregs did not express intracellular granzyme B. iTregs have been shown to produce IL-10 after re-stimulation in vitro in some studies and IL-10 can function as a potent inhibitor of costimulatory molecule expression on DC Expanded iTregs failed to produce IL-10 when they were restimulated ex vivo. To more accurately address the possibility that DCs were the targets for iTreg-mediated suppression in this model, we used a three cell transfer model (Treg, Teff, and DC). We then performed the triple transfer of antigen-specific iTregs, CFSE-labeled, nave Teff, and antigen-pulsed spleen cells and gated on the transferred Teff in the spleen 4-5 days later. Marked expansion of Teff cells was observed in recipients that received antigen-pulsed splenic DC without Treg, and marked inhibition of Teff cell expansion was seen in recipients of the iTregs. The major advantage of the three cell transfer system is that it allows for careful control of all aspects of T cell activation in vivo as we can vary the ratio of Teff:Treg, and vary the strength of the TCR signal by varying the numbers of transferred DC, or the amount of antigen used to pulse the DC. This protocol also allowed us to recover the antigen-presenting DC at different time points after cell transfer. Since Teff cell proliferation was markedly inhibited, we reasoned that iTreg-mediated suppression of DC function should be observed at an early time point after cell transfer. We re-isolated the DC from the spleen 18 hours after cell transfer and tested their capacity to activate fresh nave TCR Tg T cells specific for the target peptide. DCs from animals that did not receive either Teff or iTregs were quite efficient at activating the proliferation of nave T responder cells and similar results were observed with DCs recovered from recipients that only received Teff. However, DCs recovered from animals that received both Teff and iTregs were markedly deficient in their capacity to activate nave responder cells. This result strongly suggests that the iTregs modified DC function resulting in disabling the capacity of the DCs to present antigen. We did not observe any reduction in the expression of the co-stimulatory molecules CD80/CD86, CD40, or MHC class II expression when we analyzed DCs from mice that had been co-transferred with Treg and Teff compared to DCs from mice that received Teff alone.